This project will establish proof-of-concept for a powerful and versatile implementation of live-cell assays in a true high-throughput screening (HTS) format for small-molecule drug discovery. The technological basis is fluorescence lifetime (FLT) readout of FRET between fluorescent fusion proteins. Lifetime measurement is needed in HTS to overcome the low precision of conventional fluorescence intensity measurements, which is particularly severe in live-cell assays. However, conventional lifetime technology, i.e., time-correlated single-photon counting (TCSPC), takes at least 10 seconds per sample to obtain adequate precision for HTS. Thus, whether carried out in a microplate reader or in fluorescence lifetime imaging microscopy (FLIM), TCSPC is much too slow for practical HTS. Our team has taken an entirely fresh and creative approach, which critically relies on the revolutionary NovaFluor PR fluorescence lifetime microplate reader developed by Fluorescence Innovations. NovaFluor employs Direct Waveform Recording (DWR), an exceptionally fast and precise fluorescence lifetime method recently developed in collaboration between FI and the Thomas research group at the University of Minnesota. DWR provides precision and resolution equivalent to TCSPC while dramatically increasing the speed of data acquisition. Of all the existing fluorescence lifetime methods, only DWR offers both the speed and precision needed for effective HTS. Our other breakthrough innovation is Cells-and-Wells (CNW). We simultaneously measure the response of hundreds of cells in a microplate well, after excitation with a pulsed laser, and the lifetime readout provides HTS data as fast as any intensity-based assay employing purified protein targets, but with an order of magnitude better precision and resolution. Aim 1 is to demonstrate the CNW method on two well-defined test systems, cleavage of a labeled peptide by caspase-3 and ubiquitination of -synuclein, in order to optimize procedures in cell handling, data acquisition, instrument configuration, and data reduction. Aim 2 is to develop a high-performance assay for an important protein target, SERCA, the sarco(endo)plasmic reticulum Ca-ATPase, which is key to calcium regulation in all mammalian cells, and of particular interest in heart failure therapies. The Thomas group leads the world in developing spectroscopic probes of SERCA. Aim 3 is to conduct a first-pass screening with the LOPAC library. This work will set the stage for a more comprehensive exploration of chemical space in Phase II, leading to successful commercialization of FLT technology for drug discovery. The significance of this project stems from the clear potential of FLT in live cells to revolutionize HTS, resulting in a vastly improved input into the drug discovery process. We envision our approach will enable successful drug discovery campaigns for a wide range of targets and systems that currently can only be screened by fluorescence intensity. The high potential significance of fluorescence lifetime in HTS will make this a high-impact project, even in Phase I.